Purification of proteins from a heterogeneous mixture often involves a multi-step process that makes use of the physical, chemical and electrical properties of the protein being purified. Important properties of a protein that are relevant to its purification are (a) solubility, which determines the ability of the protein to remain in solution or to precipitate out in the presence of salt; (b) charge, which is an important property relevant to ion exchange chromatography and isoelectric focusing; (c) size, which is relevant in processes involving dialysis, gel-filtration chromatography, gel electrophoresis and sedimentation velocity; (d) specific binding, which allows purification of a protein based on its binding to a ligand; and (e) ability to form complexes in the presence of other reagents, such as in antibody precipitation. Protein detection and purification has become a major focus of research activities in view of the challenges faced by researchers involved in functional genomics and proteomics.
Popular chromatographic approaches may rely on a protein's ability to bind to a molecule linked to the matrix of a column, as in affinity chromatography, on the protein's size as in size exclusion or molecular sieving or on the pH at which the protein is electrically neutral such as in chromatofocusing or may utilize hydrophobic interactions as in reverse-phase chromatography or overall charge of the protein as in ion exchange chromatography. Since there are many different types of proteins and no single method has proved equally suitable for all proteins, it has become customary to combine different approaches to maximize yield and maintain the biological activity of the purified protein. Processes that simplify the purification of proteins with reductions in processing time remain much sought after for both large and small scale purifications.
A further area in which improved protein purification processes are greatly needed is in the recovery of recombinant proteins after expression in recombinant hosts, such as E.coli. The expression step may be highly efficient but thereafter there may be problems of improper protein folding and the protein may become sequestered within the host in inclusion bodies. Proteins in inclusion bodies are insoluble, tightly packed aggregates, generally lacking biological activity. Recovery of active proteins from inclusion bodies requires solubilization, refolding and purification of the recombinant protein product. These processes are often time-consuming and require customization for the specific protein being purified.
Silicon carbide has been shown to bind negatively charged macromolecules such as DNA and RNA. It has therefore been utilized for the purification of nucleic acid macromolecules, as described in U.S. Pat. No. 6,177,278, with many applications such as genetic research, gene therapy, genetic vaccination and cosmetics.
Protein purification methods employing interactions between proteins and silicon carbide have not been previously described.